The present invention relates generally to DNA vectors useful for introducing new genetic material into mammalian and avian cells, and particularly to a new class of retroviral vectors capable of stably transforming primary mammalian cells to provide new genotypes which express heterologous DNA sequences.
Retroviral vectors provide an efficient means of gene transfer, particularly for cells which are difficult to transfect by other methods, or which are represented at a low frequency in mixed primary populations. In the replicative cycle of retroviruses, the RNA-based genomic complement of an infecting retrovirus is reverse-transcribed to provide DNA copies known as proviruses, which are stably incorporated into cellular DNA. Generally, infection by retrovirus does not kill infected cells, and a broad variety of cell types and host species are susceptible to infection in vivo and in vitro by certain amphotropic retroviruses. Coffin, in Weiss et al., eds., RNA Tumor Viruses Vol. 2 (Cold Spring Harbor Laboratory, 1985) pp 36-71, provides a review of progress in the use of retroviruses as vectors of foreign genes.
A number of genes have been expressed in retroviral constructs, principally by utilizing the transcriptional enhancer and promoter elements in the retroviral long terminal repeat (LTR). These flanking sequences provide the transcriptional signals required for expression of proviral DNA. For example, Miller et al., Mol Cell. Biol. 5:431 (1985), employed retroviral vectors containing a mutant dihydrofolate reductase (DHFR) gene under the transcriptional control of the Moloney murine leukemia virus (MoMLV) LTRs to confer increased methotrexate resistance to infected cells.
Retroviral vectors have been constructed in which inserted genes are expressed from single heterologous internal promoters. Such promoters include those from the SV40 early region, and the Herpes Simplex Virus thymidine kinase (HSVtk), mouse metallothionein and rat growth hormone genes. Internal promoters have been used to express selectable antibiotic resistance marker in infected mammalian cells. For example, Wagner et al., EMBO J. 4:663 (1985) constructed MoMLV-based vectors in which the selectable neomycin resistance gene neo was expressed under the control of an internal thymidine kinase (TK) promoter.
Preliminary experiments involving retroviral vectors incorporating more than one internal transcriptional promoter suggested that such constructions were ineffective expression vectors due :o rearrangements of proviral DNA in infected cells. These rearrangements resulted in a partial deletion of one inserted cistron. In these experiments, Emerman and Temin, J. Virol. 50:42 (1984) assembled spleen necrosis virus (SNV)-based retroviral vectors containing the herpes simplex virus thymidine kinase (TK) and mouse .alpha.-globin genes, each including its own promoter Infected cells selected for expression of the HSVtk cistron consistently contained proviruses with deletions of the .alpha.-globin promoter. However, viruses having the .alpha.-globin gene inserted without its promoter were stable during several cell passages under the same conditions. It was proposed that the deletions occurred because the active .alpha.-globin promoter prevented efficient transcription of the HSVtk cistron in this vector, perhaps by epigenetic suppression of gene expression, or via transcriptional overlap interference. These results suggested that retroviral vectors containing multiple internal promoters could not be used effectively to co-express inserted sequences in singly infected cells.
Acutely oncogenic retroviruses contain a DNA sequence, known generically as an oncogene (onc), which is capable of transforming infected cells to a cancerous or malignant phenotype. Known retroviral oncogenes are not required for normal viral replication but appear to have evolved by transduction of normal cellular genes, known as cellular proto-oncogenes, into the viral genome. Under the control of a strong retroviral promoter, or by mutation or rearrangement, cellular proto-oncogenes acquire transforming capacity. Approximately sixty oncogenes are known, which form several principal genera on the basis of sequence homology and presumed function. In general, expression of a single oncogene is insufficient for the full transformation of primary mammalian cells. Coinjection strategies employing separate retroviruses, each bearing a single oncogene, have previously been employed to achieve transformation of Primary mammalian cells. See, e.g , Schwartz et al., Mol. Cell. Biol. 6:3221 (1986), Land et al., Nature 304:596 (1983); Land et al., Mol. Cell. Biol. 6:1917 (1986). However, this approach is not practical with mixed primary populations if the observed infection frequency of the target cell is low. Construction and use of single retroviral vectors comprising two oncogenes under the control of independent transcriptional promoters has not been reported.
The present invention resulted from attempts to create a model system in which oncogene combinations capable of immortalizing primary lymphoid or hematopietic cells could be identified. In this work, retroviral vectors were constructed which expressed two oncogenes under independent transcriptional control. A drug-resistance marker under the control of a third promoter also was incorporated into the vectors to enable selection of infected cells expressing proviral DNA. The resulting vectors transformed taret cells to a drug resistant phenotype with high efficiency, while co-expressing the oncogene products. RetroviraI vectors capable of independently expressing three or more distinct cistrons represent valuable gene transfer vehicles for transfection of primary mammalian cells.